CN113046412B - High-safety non-inactivated virus preservation solution and preparation method thereof - Google Patents
High-safety non-inactivated virus preservation solution and preparation method thereof Download PDFInfo
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Abstract
The invention provides a high-safety non-inactivated virus preservation solution and a preparation method thereof, wherein the preservation solution comprises hanks base solution, an antibiotic agent, a protein protective agent, a cryoprotectant and a nutrient agent, the protein protective agent comprises bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectant comprises sodium alginate, polyethylene glycol and glucose, the nutrient agent comprises asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agent comprises gentamycin, ancillin sodium and amikacin. The effect of bacteria and fungi on viruses is inhibited by antibiotic agents, the protein shells of the viruses are protected by protein protective agents, the protective solution can be stabilized at low temperature by freezing protective agents, and the viruses can survive in the protective solution by nutritional agents.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a high-safety non-inactivated virus preservation solution and a preparation method thereof.
Background
The virus preservation solution is used for collecting, preserving and transporting common virus samples such as novel coronavirus, influenza virus, hand-foot-and-mouth virus and the like, a liquid for protecting a detected substance of the virus is immersed in a sampling tube, a throat swab, a nose swab or a specific part tissue sample can be collected, and the stored sample can be used for subsequent clinical experiments such as nucleic acid extraction or purification.
Now, a plurality of non-inactivated virus preservation solutions have been developed, and through a great number of searches and references, it is found that the existing preservation solutions are as disclosed in the publication numbers KR1020100015613A, KR1020170095270A and KR1020100049506A, and the nucleic acid stabilizing buffer solution provided by the preservation solutions is transported and preserved at low temperature by using a non-ionic surfactant and temperate cells, so that the virus nucleic acid is effectively prevented from being degraded, and the detection false negative result is reduced. However, the virus preserved by the protective solution has stronger activity, and can cause virus infection if being treated improperly in the operation process.
Disclosure of Invention
The invention aims to provide a non-inactivated virus preservation solution with high safety against the defects.
In order to overcome the defects of the prior art, the invention adopts the following technical scheme:
the non-inactivated virus preservation solution with high safety is characterized by comprising hanks base solution, antibiotic agents, protein protective agents, cryoprotectants and nutritional agents, wherein the protein protective agents comprise bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectants comprise sodium alginate, polyethylene glycol and glucose, the nutritional agents comprise asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agents comprise gentamicin, mecillinam sodium and amikacin;
further, the weight ratio of each substance in the non-inactivated virus preservation solution is as follows: 100 parts of hanks base solution, 4 parts of antibiotic medicament, 3 parts of protein protective agent, 1 part of cryoprotectant and 1 part of nutrient;
further, the weight ratio of each substance in the protein protective agent is as follows: 10 parts of bovine serum albumin, 8 parts of mannitol, 2 parts of glycerol, 6 parts of butanediol, 3 parts of sodium chloride, 3 parts of sodium benzoate and 100 parts of water;
further, the weight ratio of each substance in the cryoprotectant is as follows: 15 parts of sodium alginate, 9 parts of polyethylene glycol, 9 parts of glucose and 100 parts of water;
further, the weight ratio of each substance in the nutrient is as follows: 3 parts of asparagine, 3 parts of glutamine, 3 parts of threonine, 2 parts of aspartic acid, 2 parts of valine, 2 parts of leucine, 1 part of isoleucine and 100 parts of water;
further, the antibiotic medicament comprises the following substances in parts by weight: 5 parts of gentamicin, 2 parts of cloxacillin sodium, 4 parts of amikacin and 50 parts of water.
The method for preparing the non-inactivated virus preservation solution comprises the following steps:
preparing an antibiotic medicament: taking gentamicin, ampicillin sodium and amikacin according to the proportion, dissolving the gentamicin, the ampicillin sodium and the amikacin in water, uniformly stirring, then carrying out autoclaving at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a protein protective agent: weighing bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage;
preparing a cryoprotectant: weighing sodium alginate, polyethylene glycol and glucose according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a nutritional agent: weighing asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine according to the proportion, dissolving in water, uniformly stirring, carrying out high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
adding the prepared antibiotic agents, protein protective agents, cryoprotectants and nutritional agents into the hanks base solution in corresponding amounts respectively, uniformly stirring, adjusting the pH value of the mixed solution to 4.5-5.2, packaging and storing in an environment at-10 ℃.
The beneficial effects obtained by the invention are as follows:
the addition of the sodium ampicillin in the antibiotic agent strengthens the inhibition effect on bacteria and fungi, the addition of the mannitol in the protein protective agent makes the adhesion of the bovine serum albumin in the protective solution covering the virus protein shell more stable, the virus is protected from being decomposed, the infectivity of the virus is greatly reduced, and the safety of the virus preservation solution in use is improved.
Drawings
The invention will be further understood from the following description in conjunction with the accompanying drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the embodiments. Like reference numerals designate corresponding parts throughout the different views.
FIG. 1 is a table of data of infectivity control experiment of non-inactivated virus preservation solution.
FIG. 2 is a schematic view showing a flow of preparation of a non-inactivated virus preservation solution.
FIG. 3 is a schematic diagram showing the ratio of each substance of the protein protectant.
Fig. 4 is a schematic diagram of the ratio of the antibiotic agents.
FIG. 5 is a schematic view of a cartridge vibrator.
Fig. 6 is a schematic view of the vibration principle of the vibrator.
Detailed Description
In order to make the objects and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following embodiments; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Other systems, methods, and/or features of the present embodiments will become apparent to those skilled in the art upon review of the following detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims. Additional features of the disclosed embodiments are described in, and will be apparent from, the detailed description that follows.
The same or similar reference numerals in the drawings of the embodiments of the present invention correspond to the same or similar components; in the description of the present invention, it should be understood that if there is an orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", etc., based on the orientation or positional relationship shown in the drawings, it is only for convenience of description and simplification of description, but it is not indicated or implied that the device or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and are not to be construed as limitations of the present patent, and specific meanings of the terms may be understood by those skilled in the art according to specific situations.
The first embodiment.
The non-inactivated virus preservation solution with high safety is characterized by comprising hanks base solution, antibiotic agents, protein protective agents, cryoprotectants and nutritional agents, wherein the protein protective agents comprise bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectants comprise sodium alginate, polyethylene glycol and glucose, the nutritional agents comprise asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agents comprise gentamicin, mecillinam sodium and amikacin;
the weight ratio of each substance in the non-inactivated virus preservation solution is as follows: 100 parts of hanks base solution, 4 parts of antibiotic medicament, 3 parts of protein protective agent, 1 part of cryoprotectant and 1 part of nutrient;
the weight ratio of each substance in the protein protective agent is as follows: 10 parts of bovine serum albumin, 8 parts of mannitol, 2 parts of glycerol, 6 parts of butanediol, 3 parts of sodium chloride, 3 parts of sodium benzoate and 100 parts of water;
the cryoprotectant comprises the following substances in parts by weight: 15 parts of sodium alginate, 9 parts of polyethylene glycol, 9 parts of glucose and 100 parts of water;
the weight ratio of each substance in the nutrient is as follows: 3 parts of asparagine, 3 parts of glutamine, 3 parts of threonine, 2 parts of aspartic acid, 2 parts of valine, 2 parts of leucine, 1 part of isoleucine and 100 parts of water;
the antibiotic preparation comprises the following substances in parts by weight: 5 parts of gentamicin, 2 parts of cloxacillin sodium, 4 parts of amikacin and 50 parts of water.
Example two.
The non-inactivated virus preservation solution with high safety is characterized by comprising hanks base solution, antibiotic agents, protein protective agents, cryoprotectants and nutritional agents, wherein the protein protective agents comprise bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectants comprise sodium alginate, polyethylene glycol and glucose, the nutritional agents comprise asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agents comprise gentamicin, mecillinam sodium and amikacin;
the weight ratio of each substance in the non-inactivated virus preservation solution is as follows: 100 parts of hanks base solution, 4 parts of antibiotic medicament, 3 parts of protein protective agent, 1 part of cryoprotectant and 1 part of nutrient;
the weight ratio of each substance in the protein protective agent is as follows: 10 parts of bovine serum albumin, 8 parts of mannitol, 2 parts of glycerol, 6 parts of butanediol, 3 parts of sodium chloride, 3 parts of sodium benzoate and 100 parts of water;
the cryoprotectant comprises the following substances in parts by weight: 15 parts of sodium alginate, 9 parts of polyethylene glycol, 9 parts of glucose and 100 parts of water;
the weight ratio of each substance in the nutrient is as follows: 3 parts of asparagine, 3 parts of glutamine, 3 parts of threonine, 2 parts of aspartic acid, 2 parts of valine, 2 parts of leucine, 1 part of isoleucine and 100 parts of water;
the antibiotic preparation comprises the following substances in parts by weight: 5 parts of gentamicin, 2 parts of cloxacillin sodium, 4 parts of amikacin and 50 parts of water;
the method for preparing the non-inactivated virus preservation solution comprises the following steps:
preparing an antibiotic medicament: taking gentamicin, ampicillin sodium and amikacin according to the proportion, dissolving the gentamicin, the ampicillin sodium and the amikacin in water, uniformly stirring, then carrying out autoclaving at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a protein protective agent: weighing bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage;
preparing a cryoprotectant: weighing sodium alginate, polyethylene glycol and glucose according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a nutritional agent: weighing asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine according to the proportion, dissolving in water, uniformly stirring, carrying out high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
adding the prepared antibiotic agents, protein protective agents, cryoprotectants and nutritional agents into hanks base liquid in corresponding amounts respectively, uniformly stirring, adjusting the pH value of the mixed solution to 4.5-5.2, packaging and storing in an environment at-10 ℃;
the prepared non-inactivated virus preservation solution and the common non-inactivated virus preservation solution are used for respectively preserving the same influenza virus, the infectivity of the virus preservation solution is tested at different stages, the experiment is divided into 6 groups, 3 groups of the prepared non-inactivated virus preservation solution preserve the influenza virus, the other 3 groups of the common non-inactivated virus preservation solution preserve the influenza virus, the influenza virus is preserved in an environment of 70 ℃ below zero, the test mode is that equal amount of sample solution is extracted from the preservation solution and added into a cell culture dish, the preservation rate of complete cells in the cell culture dish is detected, the detection result is shown in figure 1, and the virus in the prepared non-inactivated virus preservation solution has lower infectivity to the cells in the cell culture dish as can be seen from the table.
Example three.
The non-inactivated virus preservation solution with high safety is characterized by comprising hanks base solution, antibiotic agents, protein protective agents, cryoprotectants and nutritional agents, wherein the protein protective agents comprise bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectants comprise sodium alginate, polyethylene glycol and glucose, the nutritional agents comprise asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agents comprise gentamicin, mecillinam sodium and amikacin;
the weight ratio of each substance in the non-inactivated virus preservation solution is as follows: 100 parts of hanks base solution, 4 parts of antibiotic medicament, 3 parts of protein protective agent, 1 part of cryoprotectant and 1 part of nutrient;
the weight ratio of each substance in the protein protective agent is as follows: 10 parts of bovine serum albumin, 8 parts of mannitol, 2 parts of glycerol, 6 parts of butanediol, 3 parts of sodium chloride, 3 parts of sodium benzoate and 100 parts of water;
the cryoprotectant comprises the following substances in parts by weight: 15 parts of sodium alginate, 9 parts of polyethylene glycol, 9 parts of glucose and 100 parts of water;
the weight ratio of each substance in the nutrient is as follows: 3 parts of asparagine, 3 parts of glutamine, 3 parts of threonine, 2 parts of aspartic acid, 2 parts of valine, 2 parts of leucine, 1 part of isoleucine and 100 parts of water;
the antibiotic preparation comprises the following substances in parts by weight: 5 parts of gentamicin, 2 parts of cloxacillin sodium, 4 parts of amikacin and 50 parts of water;
the method for preparing the non-inactivated virus preservation solution comprises the following steps:
preparing an antibiotic medicament: taking gentamicin, ampicillin sodium and amikacin according to the proportion, dissolving the gentamicin, the ampicillin sodium and the amikacin in water, uniformly stirring, then carrying out autoclaving at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a protein protective agent: weighing bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage;
preparing a cryoprotectant: weighing sodium alginate, polyethylene glycol and glucose according to the proportion, dissolving in water, stirring uniformly, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
preparing a nutritional agent: weighing asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine according to the proportion, dissolving in water, uniformly stirring, carrying out high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use;
adding the prepared antibiotic agents, protein protective agents, cryoprotectants and nutritional agents into hanks base liquid in corresponding amounts respectively, uniformly stirring, adjusting the pH value of the mixed solution to 4.5-5.2, packaging and storing in an environment at-10 ℃;
respectively storing the same influenza virus by using the prepared non-inactivated virus preservation solution and the common non-inactivated virus preservation solution, testing the infectivity of the virus preservation solution at different stages, wherein the experiment is divided into 6 groups, 3 groups store the influenza virus by using the prepared non-inactivated virus preservation solution, and the other 3 groups store the influenza virus by using the common non-inactivated virus preservation solution, and the influenza viruses are stored in an environment at-70 ℃;
the device for preparing the virus preservation solution comprises a preparation cylinder, a high-pressure sterilization cylinder, a cooling device and a preservation cylinder;
the configuration cylinder is arranged in a vibrator, the vibrator comprises a front vertical plate and a rear vertical plate, a cylindrical panel is arranged between the vertical plates, the vertical plates are connected with the cylindrical panel through springs, a circle of spongy cushion is arranged on the inner side of the cylindrical panel, the inner diameter of the cylindrical panel is slightly larger than the outer diameter of the configuration cylinder, the spongy cushion can be extruded when the configuration cylinder is arranged in the vibrator, the spongy cushion plays roles of buffering and clamping, a clamping piece is arranged at the bottom of the cylindrical panel and is used for clamping the bottom of the configuration cylinder to prevent the configuration cylinder from sliding up and down in the cylindrical panel, a push plate is arranged between the cylindrical panel and one of the vertical plates, the push plate is arc-shaped and is matched with the cylindrical panel, a buffer cushion is arranged on one side of the push plate facing the cylindrical panel, and the other side of the push plate is connected with a connecting rod, the other end of the connecting rod is connected with the cam, the cam is connected with an output shaft of the motor, one end of the connecting rod, which is in contact with the cam, is spherical so as to reduce the friction force between the connecting rod and the cam, the connecting rod penetrates through the vertical plate, a limiting block is arranged on the vertical plate and is used for fixing the direction of the connecting rod, when the motor works, the connecting rod moves back and forth under the action of the cam, the configuration cylinder moves towards one direction together with the cylindrical panel under the thrust action of the push plate and then moves towards the opposite direction under the action of the elastic force of the spring, the oscillation effect is continuously and repeatedly realized, so that the solution in the configuration cylinder is quickly and uniformly mixed, an openable opening is arranged at the bottom of the configuration cylinder, and the opening is communicated with the high-pressure sterilization bin through a hose;
a high pressure sterilization section of thick bamboo is including disinfecting the storehouse, it is equipped with a transfer line and safe relief valve to disinfect the storehouse top, it is equipped with the drain pipe to disinfect the storehouse below, the transfer line with a configuration section of thick bamboo links to each other, the drain pipe with preserve a section of thick bamboo and link to each other, the transfer line with be equipped with the good switch of seal on the drain pipe, work as drain pipe, drain pipe and safe relief valve all are in when closed condition, it is a enclosure space to disinfect the storehouse, still be equipped with the electric heating pipeline on the storehouse outer wall of disinfecting, the electric heating pipeline is connected with the PLC controller, the PLC controller is used for control the heating power of electric heating pipeline, still be equipped with temperature-sensing ware and atmospheric pressure inductor on the storehouse inner wall of disinfecting, the temperature-sensing ware with the atmospheric pressure inductor is through setting up circuit on the storehouse wall of disinfecting with the PLC controller links to each other, the PLC controller will receive in real time the temperature-time with the temperature and atmospheric pressure that the atmospheric pressure inductor reported Information, the opening state of the safety relief valve is controlled according to the temperature and air pressure information, the sterilization bin is kept at a preset temperature and air pressure for sterilization, a safety cover is arranged on the outer side of the sterilization bin, a control panel is arranged on the safety cover and communicated with the PLC, the temperature, the air pressure and the time for sterilization can be set through the control panel, a heat insulation protective layer is arranged in the safety cover, a heat insulation protective pad is arranged in the heat insulation protective layer, the heat insulation protective layer transmits isolated heat from the sterilization bin to the outside of the safety cover, the safety is ensured, the utilization rate of energy is also ensured, a power supply component for providing electric energy for the electric heating pipeline is arranged between the sterilization bin and the safety cover, the PLC is also provided with a self-protection mechanism, and when the temperature detected by the temperature sensor or the air pressure detected by the air pressure sensor exceeds a threshold value, automatically cutting off a power supply, arranging a condensing pipe between the sterilizing bin and the safety cover, and starting the condensing pipe to perform primary cooling treatment on the sterilizing bin after high-pressure and high-temperature sterilization is finished;
the cooling device is a cup body, the preservation cylinder is arranged in the cup body, the cup wall of the cup body comprises an inner layer and an outer layer, a spiral pipeline is arranged in the cup wall of the inner layer, a coolant is filled in the spiral pipeline, a vertical pipeline is arranged on the cup wall of the outer layer, the upper part of the vertical pipeline is communicated with the upper part of the spiral pipeline, a micro liquid pump is arranged on the vertical pipeline, a heat exchange device is arranged at the bottom of the cup body, the lower part of the vertical pipeline is communicated with the lower part of the spiral pipeline through the heat exchange device to form a closed loop, the coolant slowly descends in the spiral pipeline to absorb a large amount of heat in the preservation cylinder, after a large amount of heat is released by the heat exchange device, the coolant is ascended to the upper part of the spiral pipeline through the vertical pipeline under the action of the micro liquid pump, the effect of reducing the temperature of liquid in the preservation cylinder is continuously and repeatedly achieved, and the heat exchange device comprises a serpentine pipeline, two ends of the serpentine pipeline are respectively communicated with the vertical pipeline and the spiral pipeline, the serpentine pipeline is arranged in the heat exchange pool, a steam recovery cavity is arranged above the heat exchange pool, a compression mechanism is arranged in the steam recovery cavity, radiating fins are arranged outside the steam recovery cavity and are communicated with the inside of the steam recovery cavity through heat guide rods, when the liquid in the heat exchange pool absorbs heat and evaporates to the evaporation recovery cavity and is changed back to liquid state again under the compression action of the compression mechanism, the heat released in the compression process is transferred to the radiating fins through the heat conducting rods and is diffused into the air, the steam recovery cavity is provided with a cavity door, the cavity door is in a closed state in the compression process, a plurality of small holes which can be opened and closed are arranged on the cavity door, after the compression is finished, the small hole is opened, and the liquid formed by the compression falls into the heat exchange pool through the small hole;
the invention also designs a test tube for storing the virus preservation solution, which comprises a test tube body, a sealing cap and an oil storage bag, wherein the sealing cap is provided with internal threads, the upper end of the test tube body is provided with external threads, the sealing cap is screwed on the upper end of the test tube body, the oil storage bag is arranged on the inner wall of the test tube body, when the sealing cap is screwed down, the leakage of the liquid in the test tube can be prevented, an internal stirring rod and a fixed sleeve are arranged in the sealing cap, the vertical part of the fixed sleeve is fixed on the sealing cap, the top end of the sealing cap is provided with an opening which is convenient for extruding the stirring rod to press down, the diameter of the opening is larger than that of the stirring rod and smaller than that of the fixed sleeve, the inner wall of the upper end of the stirring rod is provided with a pressing-down separation blade, one side of the upper end of the stirring rod is provided with a sharp-thorn body, when the sealing cap is screwed down, the sharp-thorn body can be matched with the oil storage bag, after the stirring rod is pressed down, the sharp pricks can pierce the oil storage bag, and oily liquid in the oil storage bag can cover the upper part of the virus preservation liquid to prevent the virus from leaking.
Although the invention has been described above with reference to various embodiments, it should be understood that many changes and modifications may be made without departing from the scope of the invention. That is, the methods, systems, and devices discussed above are examples. Various configurations may omit, substitute, or add various procedures or components as appropriate. For example, in alternative configurations, the methods may be performed in an order different than that described, and/or various components may be added, omitted, and/or combined. Moreover, features described with respect to certain configurations may be combined in various other configurations, as different aspects and elements of the configurations may be combined in a similar manner. Further, elements therein may be updated as technology evolves, i.e., many elements are examples and do not limit the scope of the disclosure or claims.
Specific details are given in the description to provide a thorough understanding of the exemplary configurations including implementations. However, configurations may be practiced without these specific details, for example, well-known circuits, processes, algorithms, structures, and techniques have been shown without unnecessary detail in order to avoid obscuring the configurations. This description provides example configurations only, and does not limit the scope, applicability, or configuration of the claims. Rather, the foregoing description of the configurations will provide those skilled in the art with an enabling description for implementing the described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.
In conclusion, it is intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that these examples are illustrative only and are not intended to limit the scope of the invention. After reading the description of the invention, the skilled person can make various changes or modifications to the invention, and these equivalent changes and modifications also fall into the scope of the invention defined by the claims.
Claims (2)
1. The non-inactivated virus preservation solution with high safety is characterized by comprising hanks base solution, antibiotic agents, protein protective agents, cryoprotectants and nutritional agents, wherein the protein protective agents comprise bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate, the cryoprotectants comprise sodium alginate, polyethylene glycol and glucose, the nutritional agents comprise asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine, and the antibiotic agents comprise gentamicin, mecillinam sodium and amikacin;
the weight ratio of each substance in the non-inactivated virus preservation solution is as follows: 100 parts of hanks base solution, 4 parts of antibiotic medicament, 3 parts of protein protective agent, 1 part of cryoprotectant and 1 part of nutrient;
the weight ratio of each substance in the protein protective agent is as follows: 10 parts of bovine serum albumin, 8 parts of mannitol, 2 parts of glycerol, 6 parts of butanediol, 3 parts of sodium chloride, 3 parts of sodium benzoate and 100 parts of water;
the cryoprotectant comprises the following substances in parts by weight: 15 parts of sodium alginate, 9 parts of polyethylene glycol, 9 parts of glucose and 100 parts of water;
the weight ratio of each substance in the nutrient is as follows: 3 parts of asparagine, 3 parts of glutamine, 3 parts of threonine, 2 parts of aspartic acid, 2 parts of valine, 2 parts of leucine, 1 part of isoleucine and 100 parts of water;
the antibiotic preparation comprises the following substances in parts by weight: 5 parts of gentamicin, 2 parts of cloxacillin sodium, 4 parts of amikacin and 50 parts of water;
the device for preparing the virus preservation solution comprises a preparation cylinder, a high-pressure sterilization cylinder, a cooling device and a preservation cylinder; the configuration cylinder is arranged in a vibrator, the vibrator comprises a front vertical plate and a rear vertical plate, a cylindrical panel is arranged between the vertical plates, the vertical plates are connected with the cylindrical panel through springs, a circle of spongy cushion is arranged on the inner side of the cylindrical panel, the inner diameter of the cylindrical panel is slightly larger than the outer diameter of the configuration cylinder, the spongy cushion can be extruded when the configuration cylinder is arranged in the vibrator, the spongy cushion plays roles of buffering and clamping, a clamping piece is arranged at the bottom of the cylindrical panel and is used for clamping the bottom of the configuration cylinder to prevent the configuration cylinder from sliding up and down in the cylindrical panel, a push plate is arranged between the cylindrical panel and one of the vertical plates, the push plate is arc-shaped and is matched with the cylindrical panel, a buffer cushion is arranged on one side of the push plate facing the cylindrical panel, and the other side of the push plate is connected with a connecting rod, the other end of the connecting rod is connected with the cam, the cam is connected with an output shaft of the motor, one end of the connecting rod, which is in contact with the cam, is spherical so as to reduce the friction force between the connecting rod and the cam, the connecting rod penetrates through the vertical plate, a limiting block is arranged on the vertical plate and is used for fixing the direction of the connecting rod, when the motor works, the connecting rod moves back and forth under the action of the cam, the configuration cylinder moves towards one direction together with the cylindrical panel under the thrust action of the push plate and then moves towards the opposite direction under the action of the elastic force of the spring, the oscillation effect is continuously and repeatedly realized, so that the solution in the configuration cylinder is quickly and uniformly mixed, an openable opening is arranged at the bottom of the configuration cylinder, and the opening is communicated with the high-pressure sterilization bin through a hose; a high pressure sterilization section of thick bamboo is including disinfecting the storehouse, it is equipped with a transfer line and safe relief valve to disinfect the storehouse top, it is equipped with the drain pipe to disinfect the storehouse below, the transfer line with a configuration section of thick bamboo links to each other, the drain pipe with preserve a section of thick bamboo and link to each other, the transfer line with be equipped with the good switch of seal on the drain pipe, work as drain pipe, drain pipe and safe relief valve all are in when closed condition, it is a enclosure space to disinfect the storehouse, still be equipped with the electric heating pipeline on the storehouse outer wall of disinfecting, the electric heating pipeline is connected with the PLC controller, the PLC controller is used for control the heating power of electric heating pipeline, still be equipped with temperature-sensing ware and atmospheric pressure inductor on the storehouse inner wall of disinfecting, the temperature-sensing ware with the atmospheric pressure inductor is through setting up circuit on the storehouse wall of disinfecting with the PLC controller links to each other, the PLC controller will receive in real time the temperature-time with the temperature and atmospheric pressure that the atmospheric pressure inductor reported Information, the opening state of the safety relief valve is controlled according to the temperature and air pressure information, the sterilization bin is kept at a preset temperature and air pressure for sterilization, a safety cover is arranged on the outer side of the sterilization bin, a control panel is arranged on the safety cover and communicated with the PLC, the temperature, the air pressure and the time for sterilization can be set through the control panel, a heat insulation protective layer is arranged in the safety cover, a heat insulation protective pad is arranged in the heat insulation protective layer, the heat insulation protective layer transmits isolated heat from the sterilization bin to the outside of the safety cover, the safety is ensured, the utilization rate of energy is also ensured, a power supply component for providing electric energy for the electric heating pipeline is arranged between the sterilization bin and the safety cover, the PLC is also provided with a self-protection mechanism, and when the temperature detected by the temperature sensor or the air pressure detected by the air pressure sensor exceeds a threshold value, automatically cutting off a power supply, arranging a condensing pipe between the sterilizing bin and the safety cover, and starting the condensing pipe to perform primary cooling treatment on the sterilizing bin after high-pressure and high-temperature sterilization is finished; the cooling device is a cup body, the preservation cylinder is arranged in the cup body, the cup wall of the cup body comprises an inner layer and an outer layer, a spiral pipeline is arranged in the cup wall of the inner layer, a coolant is filled in the spiral pipeline, a vertical pipeline is arranged on the cup wall of the outer layer, the upper part of the vertical pipeline is communicated with the upper part of the spiral pipeline, a micro liquid pump is arranged on the vertical pipeline, a heat exchange device is arranged at the bottom of the cup body, the lower part of the vertical pipeline is communicated with the lower part of the spiral pipeline through the heat exchange device to form a closed loop, the coolant slowly descends in the spiral pipeline to absorb a large amount of heat in the preservation cylinder, after a large amount of heat is released by the heat exchange device, the coolant is ascended to the upper part of the spiral pipeline through the vertical pipeline under the action of the micro liquid pump, the effect of reducing the temperature of liquid in the preservation cylinder is continuously and repeatedly achieved, and the heat exchange device comprises a serpentine pipeline, two ends of the serpentine pipeline are respectively communicated with the vertical pipeline and the spiral pipeline, the serpentine pipeline is arranged in the heat exchange pool, a steam recovery cavity is arranged above the heat exchange pool, a compression mechanism is arranged in the steam recovery cavity, radiating fins are arranged outside the steam recovery cavity and are communicated with the inside of the steam recovery cavity through heat guide rods, when the liquid in the heat exchange pool absorbs heat and evaporates to the evaporation recovery cavity and is changed back to liquid state again under the compression action of the compression mechanism, the heat released in the compression process is transferred to the radiating fins through the heat conducting rods and is diffused into the air, the steam recovery cavity is provided with a cavity door, the cavity door is in a closed state in the compression process, a plurality of small holes which can be opened and closed are arranged on the cavity door, after the compression is finished, the small hole is opened, and the liquid formed by the compression falls into the heat exchange pool through the small hole;
the device for preparing the virus preservation solution further comprises a test tube for storing the virus preservation solution, and comprises a test tube body, a sealing cap and an oil storage bag, wherein the sealing cap is provided with an internal thread, the upper end of the test tube body is provided with an external thread, the sealing cap is in threaded connection with the upper end of the test tube body, the oil storage bag is arranged on the inner wall of the test tube body, when the sealing cap is screwed down, the liquid in the test tube can be prevented from leaking, an inner stirring rod and a fixing sleeve are arranged in the sealing cap, the vertical part of the fixing sleeve is fixed on the sealing cap, the top end of the sealing cap is provided with an opening which is convenient for extruding the stirring rod to press down, the diameter of the opening is larger than that of the stirring rod and smaller than that of the fixing sleeve, a pressing-down separation blade is arranged on the inner wall of the upper end of the stirring rod, a sharp-prick body is arranged on one side of the upper end of the stirring rod, when the sealing cap is screwed down, the sharp thorns can pierce the oil storage bag after being pressed down and the oil liquid in the oil storage bag can cover the upper part of the virus preservation liquid to prevent the virus from leaking.
2. A method for preparing the highly safe non-inactivated virus preservation solution according to claim 1, comprising the steps of: preparing an antibiotic medicament: taking gentamicin, ampicillin sodium and amikacin according to the proportion of each substance in the antibiotic medicament, dissolving the gentamicin, the ampicillin sodium and the amikacin in water, uniformly stirring, carrying out autoclaving at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use; preparing a protein protective agent: weighing bovine serum albumin, mannitol, glycerol, butanediol, sodium chloride and sodium benzoate according to the weight proportion of the substances in the protein protectant, dissolving the bovine serum albumin, the mannitol, the glycerol, the butanediol, the sodium chloride and the sodium benzoate in water, uniformly stirring, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage; preparing a cryoprotectant: weighing sodium alginate, polyethylene glycol and glucose according to the weight ratio of the substances in the cryoprotectant, dissolving the sodium alginate, the polyethylene glycol and the glucose in water, uniformly stirring, performing high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use; preparing a nutritional agent: weighing asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine according to the weight proportion of the substances in the nutrient, dissolving the asparagine, glutamine, threonine, aspartic acid, valine, leucine and isoleucine in water, uniformly stirring, carrying out high-pressure sterilization at 120 ℃ for 15 minutes, and then cooling to 10 ℃ for storage for later use; adding the prepared antibiotic agents, protein protective agents, cryoprotectants and nutritional agents into the hanks base solution in corresponding amounts respectively, uniformly stirring, adjusting the pH value of the mixed solution to 4.5-5.2, packaging and storing in an environment at-10 ℃.
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